The global burden of tuberculosis (TB) is enormous. Drug-resistant TB, which is less effectively treated by current anti-TB therapy than drug-susceptible disease, is also a global concern. The most difficult-to-treat forms of drug-resistant TB are multidrug-resistant TB (MDR-TB; defined as resistance to at least isoniazid and rifampin) and extensively drug-resistant TB (XDR-TB; defined as MDR-TB with additional resistance to a fluoroquinolone (FQ) and an injectable agent (e.g., capreomycin)). The FQ class of antibiotics has potent activity against M. tuberculosis, could help shorten the treatment duration for drug-susceptible TB, and is very effective in treating MDR-TB. However, FQ resistance has become an increasing concern. FQ resistance can be conferred by genetic mutations in their target, DNA gyrase. However, approximately 45% of phenotypically FQ-resistant M. tuberculosis isolates do not have the commonly-described resistance mutations in DNA gyrase. This will result in unacceptably low sensitivity of rapid molecular diagnostic tests, which currently utilize the common DNA gyrase resistance mutations. Recent work has demonstrated the importance of efflux pumps in FQ resistance in M. tuberculosis. Efflux pumps may either be the sole mechanism of resistance, or a gateway to other resistance mechanisms, including DNA gyrase mutations. In addition, efflux pumps may generate cross-resistance or decrease susceptibility to other TB drugs. The goal of this proposal is to identify and characterize the efflux pumps involved in FQ resistance in M. tuberculosis. It will also characterize the genes that may modify efflux pumps, including transcription factors and phosphate transporters. We will also determine whether HIV infection enables the acquisition of efflux pump-mediated FQ resistance in M. tuberculosis-both FQ mono-resistant M. tuberculosis, and FQ-resistant strains that are MDR- or XDR-TB. The extent to which efflux pumps in FQ-resistant M. tuberculosis decrease susceptibility to other anti-TB drugs, including new agents, will also be assessed.